title: Oligonucleotides labeled with single fluorophores as sensors for deoxynucleotide triphosphate binding by DNA polymerases.
abstract: Oligonucleotides labeled with a single fluorophore (fluorescein or tetramethylrhodamine) have been used previously as fluorogenic substrates for a number of DNA modifying enzymes. Here, it is shown that such molecules can be used as fluorogenic probes to detect the template-dependent binding of deoxynucleotide triphosphates by DNA polymerases. Two polymerases were used in this work: the Klenow fragment of the Escherichia coli DNA polymerase I and the Bacillus stearothermophilus polymerase, Bst. When complexes of these polymerases with dye-labeled hairpin-type oligonucleotides were mixed with various deoxynucleotide triphosphates in the presence of Sr²⁺ as the divalent metal cation, the formation of ternary DNA-polymerase-dNTP complexes was detected by concentration-dependent changes in the fluorescence intensities of the dyes. Fluorescein- and tetramethylrhodamine-labeled probes of identical sequences responded differently to the two polymerases. With Bst polymerase, the fluorescence intensities of all probes increased with the next correct dNTP; with Klenow polymerase, tetramethylrhodamine-labeled probes increased their fluorescence, but the intensity of fluorescein-labeled probes decreased on formation of ternary complexes with the correct incoming nucleotides. The use of Sr²⁺ as the divalent metal ion allowed the formation of catalytically inactive ternary complexes and obviated the need for using 2',3'-dideoxy-terminated oligonucleotides as would have been needed in the case of Mg²⁺ as the metal ion.
authors.affiliation: Life Technologies, Carlsbad, CA 92008, USA. Electronic address: theo.nikiforov@lifetech.com.
authors.forename: Theo T
authors.initials: TT
authors.lastname: Nikiforov
mesh_terms: D001665:Binding Sites; D004256:DNA Polymerase I / Q000032:analysis / Q000378:metabolism*; D003854:Deoxyribonucleotides / Q000032:analysis / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D005456:Fluorescent Dyes / Q000032:analysis* / Q000737:chemistry*; D001411:Geobacillus stearothermophilus / Q000201:enzymology; D009841:Oligonucleotides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding
chemical_list: D003854:Deoxyribonucleotides; D005456:Fluorescent Dyes; D009841:Oligonucleotides; D004256:DNA Polymerase I
keywords: Bst polymerase; DNA polymerases; Fluorescence; Klenow fragment; dNTPs